1. Field of the Invention
The invention relates to the screening of blood or a blood product (including whole blood, hematopoietic stem cells, leukocytes, plasma, serum, red blood cells, and platelets) or a donor tissue for the presence of a clinically relevant amount of bacteria More particularly, the invention relates to the screening for the presence of a clinically relevant amount of contaminating bacteria in blood and blood products or donor tissue that will be used for transfusion or transplantation.
2. Summary of the Related Art
Transfusion of blood and blood products is a therapeutically important aspect of patient care. Transplantation of donor tissues and organs is likewise therapeutically important. The absence of clinically relevant levels of contaminating bacteria in donor blood, donor blood product, or a donor tissue or organ is a requirement necessary for the safe, therapeutic use of these donated fluids and tissues for transfusion or transplantation. For example, bacteremia (the invasion of bacteria into the blood) can be transient, continuous, or intermittent. Wagner et al. (Clin. Microbiol. Rev. 7: 290-302 (1994)) and Goldman et al. (Trans. Med. Revs. 5: 73-83 (1991)) teach that a large number of different species of bacteria have been identified in contaminated blood transfused to patients who, following transfusion, developed septicimia. Tadler et al. (J. Clin. Laboratory Analysis 2: 21-25 (1989)) teaches that although transient bacteremia is generally of little consequence, continuous or intermittent bacteremia can present life-threatening situations for several patient populations, particularly immunocompromised, neonatal, and geriatric patients. Thus, if blood contaminated with a clinically relevant amount of bacteria were to be transfused to a recipient patient, the recipient patient might suffer complications, particularly since transfusions of blood and/or blood products are often performed when the patient is undergoing major surgery, or is otherwise vulnerable.
Similarly, donor tissues and organs are preferably microbe-free to retard and, preferably, prevent rejection by the recipient.
Despite the need for a safe, microbe-free supply of blood or a blood product, no rapid, efficient method exists for detecting the presence of contaminating bacteria in blood or a blood product. Although there are methods for determining whether or not blood is infected with bacteria, most current bacterial testing is done on patients suspected of having infected blood, with the prime intent of identifying the exact micro-organism that is causing the infection, so that the appropriate antibiotic therapy can begin. In these methods, to identify the infecting bacteria, a sample of patient blood is grown in a culture media that favors the growth of the bacteria for a relatively long period of time. Eventually, the number of micro-organisms present is amplified to the point that a reasonable quantity exists and can be detected.
Although these culture-based blood testing techniques are useful for determining the particular type of bacteria that is infecting a patient""s blood, the length of the time required to perform these techniques makes them impractical to use to test donor blood and blood products or donor organs. This is because donor blood, blood products, and organs are often needed for use as transfusions or transplantations on relatively short notice. Thus, there may be no time to test the donated blood (or blood product) or organ with a culture-based technique before the donated blood or organ is needed for use.
In addition, donated organs and blood or blood products have a relatively short shelf-life due not only to a loss of function of the donated material, but also to an increase in amount of any contaminating bacteria present. Since bacteria have a rapid propagation rate, even a small amount of contaminating bacteria present in the donated blood or blood product will quickly amplify with time. For example, while donated platelets are functionally viable only 7 days post-donation, they are rarely used more than 5 days post-donation for fear of bacterial contamination that, when donated, may not have been significant but, over time, may have increased to a level that is clinically relevant. Moreover, these culture-based blood testing techniques take too long to routinely screen platelets, which have a short shelf-life (approximately 5 days post-donation), for transfusion use.
Brecher et al. (Transfusion 34(9): 750-755 (1994)) teaches another technique for detecting a particular contaminating bacteria in blood using labelled nucleic acid probes to hybridize to the genetic material of potential contaminants The probes used in these studies, however, are very limited in the number of micro-organisms that can be detected and, unfortunately, no commercially viable test has emerged from this technology. Given their complexity, these techniques are too labor-intensive and too time-costly to be routinely used to screen blood or blood products for bacterial contamination.
There is, therefore, a need for a technique for rapidly detecting the presence of a clinically relevant amount of any contaminating bacteria in donor blood or a blood product, or in a donor tissue. Ideally, antigen binding techniques could be rapidly and effectively employed. Unfortunately, Wagner, S. J. (Int. J. Med. Microbiol. Virol. Parasitol. Infect. Dis. 283(3):253-257 (1996)) teaches that no common antigenic source exists for broad based bacterial detection. Thus, there is a need for antigen binding-based techniques for detecting clinically relevant amounts of contaminating bacteria in donor blood or blood products or in donor tissues.
The invention provides rapid antigen binding-based methods for detecting clinically relevant amounts of contaminating bacteria in blood or blood products or in donor tissue, particularly donor blood or blood products or donor tissue to be transferred from one individual to another.
Accordingly, in a first aspect, the invention provides a method for screening for the presence of a clinically relevant amount of bacteria in donor blood or a donor blood product from a donor mammal for transfer to a recipient mammal comprising contacting a sample of blood or a blood product with a set of binding agents, wherein the set of binding agents comprises binding agents that specifically bind to a Gram-negative bacterial antigen and binding agents that specifically bind to a Gram-positive bacterial antigen, and determining binding of the set of binding agents to the sample, wherein binding indicates the presence of a clinically relevant amount of bacteria in the donor blood or blood product and no binding indicates the absence of a clinically relevant amount of bacteria in the donor blood or blood product.
In certain embodiments of the first aspect of the invention, the sample is treated prior to or concomitantly with contacting the sample with the set of binding agents. Preferably, the treatment exposes a binding site of the binding agent on the Gram-negative bacterial antigen or on the Gram-positive bacterial antigen. In certain embodiments of the first aspect of the invention, the donor blood or blood product determined to have an absence of a clinically relevant amount of bacteria is administered to a recipient mammal. Preferably, the donor mammal and the recipient mammal are of the same species. In certain preferred embodiments, the binding agents that specifically bind to a Gram-negative bacterial antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-negative bacterial antigen. In certain preferred embodiments, the binding agents that specifically bind to a Gram-negative bacterial antigen thereof comprise a molecule selected from the group consisting of a Limulus anti-lipopolysaccharide factor (LALF), a lipopolysaccharide binding protein (LBP), a bactericidal/permeability-increasing protein (BPI), and an antibiotic, such as polymixin or bacitracin. Preferably, the binding agents that specifically bind to a Gram-negative bacterial antigen specifically bind to the lipopolysaccharide structure of the Gram-negative bacteria. In certain preferred embodiments, the binding agents that specifically bind to a Gram-positive bacteria antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-positive bacterial antigen. In certain embodiments, the binding agents that specifically bind to a Gram-positive bacterial antigen comprise an antibiotic, wherein the antibiotic is not vancomycin. Preferably, the binding agents that specifically bind to a Gram-positive bacterial antigen specifically bind to the lipotechoic acid structure of the Gram-positive bacteria.
In various embodiments of the first aspect of the invention, the binding agents that specifically bind to a Gram-negative bacterial antigen are detectably labeled with a first reporter molecule and the binding agents that specifically bind to a Gram-positive bacterial antigen are detectably labeled with a second reporter molecule. In certain embodiments, the first reporter molecule and the second reporter molecule are the same. In another embodiment, the first reporter molecule and the second reporter molecule are not the same. Preferably, one or both of the first reporter molecule and the second reporter molecule is a molecule with enzymatic activity, a radio-labeled molecule, a fusion molecule, a fluorogenic molecule, a metal sol, a particle, a chromatic molecule, or a molecule that is specifically bound by a secondary binding agent.
In a second aspect, the invention provides method for screening for the presence of a clinically relevant amount of Gram-positive bacteria in donor blood or a blood product from a donor mammal for transfer to a recipient mammal comprising contacting a sample of the donor blood or a blood product with a set of binding agents, wherein the set of binding agents comprises binding agents that specifically bind to Gram-positive bacterial antigen, and determining binding of the set of binding agents to the sample, wherein binding indicates the presence of a clinically relevant amount of Gram-positive bacteria in the donor blood or blood product and no binding indicates the absence of a clinically relevant amount of Gram-positive bacteria in the donor blood or blood product.
In certain embodiments of the second aspect of the invention, the sample is treated prior to or concomitantly with contacting the sample with the set of binding agents. Preferably, the treatment exposes a binding site for the binding agent on the Gram-positive bacterial antigen. In certain embodiments, the blood or blood product determined to have an absence of a clinically relevant amount of Gram-positive bacteria is transferred to a recipient mammal. Preferably, the donor mammal and the recipient mammal are of the same species. In certain preferred embodiments, the binding agents that specifically bind to a Gram-positive bacterial antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-positive bacterial antigen. In certain embodiments, the binding agents that specifically bind to a Gram-positive bacterial antigen comprise an antibiotic, wherein the antibiotic is not vancomycin. Preferably, the binding agents that specifically bind to a Gram-positive bacterial antigen specifically bind to the lipotechoic acid structure of the Gram-positive bacteria.
In various embodiments of the second aspect of the invention, the binding agents that specifically bind to a Gram-positive bacterial antigen are detectably labeled with a reporter molecule. Preferably, the reporter molecule is a molecule with enzymatic activity, a radio-labeled molecule, a fusion molecule, a fluorogenic molecule, a metal sol a particle, a chromatic molecule, or a molecule that is specifically bound by a secondary binding agent.
In a third aspect, the invention provides a method for screening for the presence of a clinically relevant amount of Gram-negative bacteria in donor blood or a blood product from a donor mammal for transfer to a recipient mammal comprising contacting a sample of blood or a blood product with a set of binding agents, wherein the set of binding agents comprises binding agents that specifically bind to Gram-negative bacterial antigen, and determining binding of the set of binding agents to the sample, wherein binding indicates the presence of a clinically relevant amount of Gram-negative bacteria in the donor blood or blood product and no binding indicates the absence of a clinically relevant amount of Gram-negative bacteria in the donor blood or blood product.
In certain embodiments of the third aspect of the invention, the sample is treated prior to or concomitantly with contacting the sample with the set of binding agents. Preferably, the treatment exposes a binding site for the binding agent on the Gram-negative bacterial antigen. In certain embodiments, the donor blood or blood product determined to have an absence of a clinically relevant amount of Gram-negative bacteria is transferred to a recipient mammal. Preferably, the donor mammal and the recipient mammal are of the same species. In certain preferred embodiments, the binding agents that specifically bind to a Gram-negative bacterial antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-negative bacterial antigen. In certain preferred embodiments, the binding agents that specifically bind to a Gram-negative bacterial antigen comprise a molecule selected from the group consisting of a Limulus anti-lipopolysaccharide factor (LALF), a lipopolysaccharide binding protein (LBP), a bactericidal/permeability-increasing protein (BPI), and an antibiotic, such as polymixin or bacitracin. Preferably, the binding agents that specifically bind to a Gram-negative bacterial antigen specifically bind to the lipopolysaccharide structure of the Gram-negative bacteria.
In various embodiments of the third aspect of the invention, the binding agents that specifically bind to a Gram-negative bacterial antigen are detectably labeled with a reporter molecule. Preferably, the reporter molecule is a molecule with enzymatic activity, a radio-labeled molecule, a fusion molecule, a fluorogenic molecule, a metal sol, a particle, a chromatic molecule, or a molecule that is specifically bound by a secondary binding agent.
In a fourth aspect, the invention provides a kit for screening for the presence of a clinically relevant amount of bacteria in donor blood or a donor blood product from a donor mammal for transfer to a recipient mammal comprising a set of binding agents, wherein the set of binding agents comprises binding agents that specifically bind to Gram-negative bacterial antigen and binding agents that specifically bind to Gram-positive bacterial antigen, and a means for detecting binding of the set of binding agents to a sample of the donor blood or blood product, wherein binding indicates the presence of a clinically relevant amount of bacteria in the donor blood or blood product and no binding indicates the absence of a clinically relevant amount of bacteria in the donor blood or blood product.
In certain embodiments of the fourth aspect of the invention, the kit further comprises a means for treating the sample, wherein the treatment exposes a binding site for the binding agent on the Gram-negative bacterial antigen or on the Gram-positive bacterial antigen. In certain embodiments of the fourth aspect of the invention, the donor blood or blood product determined to have an absence of a clinically relevant amount of bacteria is transferred to a recipient mammal. Preferably, the donor mammal and the second mammal are of the same species. In certain preferred embodiments, the binding agents that specifically bind to a Gram-negative bacterial antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-negative bacterial antigen. In certain preferred embodiments, the binding agents that specifically bind to a Gram-negative bacterial antigen comprise a molecule selected from the group consisting of a Limulus anti-lipopolysaccharide factor (LALF), a lipopolysaccharide binding protein (LBP), a bactericidal/permeability-increasing protein (BPI), and an antibiotic, such as polymixin or bacitracin. Preferably, the binding agents that specifically bind to a Gram-negative bacterial antigen specifically bind to the lipopolysaccharide structure of the Gram-negative bacteria. In certain preferred embodiments, the binding agents that specifically bind to a Gram-positive bacterial antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-positive bacterial antigen. In certain embodiments, the binding agents that specifically bind to a Gram-positive bacterial antigen comprise an antibiotic, wherein the antibiotic is not vancomycin. Preferably, the binding agents that specifically bind to a Gram-positive bacterial antigen specifically bind to the lipotechoic acid structure of the Gram-positive bacteria.
In various embodiments of the fourth aspect of the invention, the binding agents that specifically bind to a Gram-negative bacterial antigen are detectably labeled with a first reporter molecule and the binding agents that specifically bind to a Gram-positive bacterial antigen are detectably labeled with a second reporter molecule. In certain embodiments, the first reporter molecule and the second reporter molecule are the same. In another embodiment, the first reporter molecule and the second reporter molecule are not the same. Preferably, one or both of the first reporter molecule and the second reporter molecule is a molecule with enzymatic activity, a radio-labeled molecule, a fusion molecule, a fluorogenic molecule, a metal sol, a particle, a chromatic molecule, or a molecule that is specifically bound by a secondary binding agent.
In a fifth aspect, the invention provides a kit for screening for the presence of a clinically relevant amount of Gram-positive bacteria in donor blood or a donor blood product from a donor mammal for transfer to a recipient mammal comprising a set of binding agents, wherein the set of binding agents comprises binding agents that specifically bind to a Gram-positive bacterial antigen, and a means for detecting binding of the set of binding agents to a sample of the blood or blood product, wherein binding indicates the presence of a clinically relevant amount of Gram-positive bacteria in the donor blood or blood product and no binding indicates the absence of a clinically relevant amount of Gram-positive bacteria in the donor blood or blood product.
In certain embodiments of the fifth aspect of the invention, the kit further comprises a means for treating the sample, wherein the treatment exposes a binding site for the binding agent on the Gram-positive bacterial antigen. In certain embodiments, the donor blood or blood product determined to have an absence of a clinically relevant amount of bacteria is transferred to a recipient mammal. Preferably, the donor mammal and the recipient mammal are of the same species. In certain preferred embodiments, the binding agents that specifically bind to a Gram-positive bacterial antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-positive bacterial antigen. In certain embodiments, the binding agents that specifically bind to a Gram-positive bacterial antigen comprise an antibiotic, wherein the antibiotic is not vancomycin. Preferably, the binding agents that specifically bind to a Gram-positive bacterial antigen specifically bind to the lipotechoic acid structure of the Gram-positive bacteria.
In various embodiments of the fifth aspect of the invention, the binding agents that specifically bind to a Gram-positive bacterial antigen are detectably labeled with a reporter molecule. Preferably, the reporter molecule is a molecule with enzymatic activity, a radio-labeled molecule, a fusion molecule, a fluorogenic molecule, a metal sol, a particle, a chromatic molecule, or a molecule that is specifically bound by a secondary binding agent.
In a sixth aspect, the invention provides a kit for screening for the presence of a clinically relevant amount of Gram-negative bacteria in donor blood or a donor blood product from a donor mammal for transfer to a recipient mammal comprising a set of binding agents, wherein the set of binding agents comprises binding agents that specifically bind to a Gram-negative bacterial antigen, and a means for detecting binding of the set of binding agents to a sample of the blood or blood product, wherein binding indicates the presence of a clinically relevant amount of Gram-negative bacteria in the donor blood or blood product and no binding indicates the absence of a clinically relevant amount of Gram-negative bacteria in the donor blood or blood product.
In certain embodiments of the sixth aspect of the invention, the kit further comprises a means for treating the sample, wherein the treatment exposes a binding site for the binding agent on the Gram-negative bacterial antigen. In certain embodiments, the donor blood or blood product determined to have an absence of a clinically relevant amount of Gram-negative bacteria is transferred to a recipient mammal. Preferably, the donor mammal and the recipient mammal are of the same species. In certain preferred embodiments, the binding agents that specifically bind to a Gram-negative bacterial antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-negative bacterial antigen. In certain preferred embodiments, the binding agents that specifically bind to a Gram-negative bacterial antigen comprise a molecule selected from the group consisting of a Limulus anti-lipopolysaccharide factor (LALF), a lipopolysaccharide binding protein (LBP), a bactericidal/permeability-increasing protein (BPI), and an antibiotic, such as polymixin or bacitracin. Preferably, the binding agents that specifically bind to a Gram-negative bacterial antigen specifically bind to the lipopolysaccharide structure of the Gram-negative bacteria.
In various embodiments of the sixth aspect of the invention, the binding agents that specifically bind to a Gram-negative bacterial antigen are detectably labeled with a reporter molecule. Preferably, the reporter molecule is a molecule with enzymatic activity, a radio-labeled molecule, a fusion molecule, a fluorogenic molecule, a metal sol, a particle, a chromatic molecule, or a molecule that is specifically bound by a secondary binding agent.
In various embodiments of the first, second, third, fourth, fifth, and sixth aspect of the invention, the blood or blood product is preferably whole blood, leukocytes, hematopoietic stem cells, platelets, red blood cells, plasma, or serum.
In a seventh aspect, the invention provides a method for screening for the presence of a clinically relevant amount of bacteria in a tissue from a mammal wherein the tissue is stored in a fluid, comprising contacting a sample of the fluid with a set of binding agents, wherein the set of binding agents comprises binding agents that specifically bind to a Gram-negative bacterial antigen and binding agents that specifically bind to a Gram-positive bacterial antigen, and determining binding of the set of binding agents to the sample, wherein binding indicates the presence of a clinically relevant amount of bacteria in the donor tissue and no binding indicates the absence of a clinically relevant amount of bacteria in the donor tissue.
In certain embodiments of the seventh aspect of the invention, the sample is treated prior to or concomitantly with contacting the sample with the set of binding agents. Preferably, the treatment exposes a binding site of the binding agent on the Gram-negative bacterial antigen or on the Gram-positive bacterial antigen. In certain embodiments of the seventh aspect of the invention, the donor tissue determined to have an absence of a clinically relevant amount of bacteria is transferred to a recipient mammal. Preferably, the donor mammal and the recipient mammal are of the same species. In certain preferred embodiments, the binding agents that specifically bind to a Gram-negative bacterial antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-negative bacterial antigen. In certain preferred embodiments, the binding agents that specifically bind to a Gram-negative bacterial antigen comprise a molecule selected from the group consisting of a Limulus anti-lipopolysaccharide factor (LALF), a lipopolysaccharide binding protein (LBP), a bactericidal/permeability-increasing protein (BPI), and an antibiotic, such as polymixin or bacitracin. Preferably, the binding agents that specifically bind to a Gram-negative bacterial antigen specifically bind to the lipopolysaccharide structure of the Gram-negative bacteria. In certain preferred embodiments, the binding agents that specifically bind to a Gram-positive bacterial antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-positive bacterial antigen. In certain embodiments, the binding agents that specifically bind to a Gram-positive bacterial antigen comprise an antibiotic, wherein the antibiotic is not vancomycin. Preferably, the binding agents that specifically bind to a Gram-positive bacterial antigen specifically bind to the lipotechoic acid structure of the Gram-positive bacteria.
In various embodiments of the seventh aspect of the invention, the binding agents that specifically bind to a Gram-negative bacterial antigen are detectably labeled with a first reporter molecule and the binding agents that specifically bind to Gram-positive bacterial antigen are detectably labeled with a second reporter molecule. In certain embodiments, the first reporter molecule and the second reporter molecule are the same. In another embodiment, the first reporter molecule and the second reporter molecule are not the same. Preferably, one or both of the first reporter molecule and the second reporter molecule is a molecule with enzymatic activity, a radio-labeled molecule, a fusion molecule, a fluorogenic molecule, a metal sol, a particle, a chromatic molecule, or a molecule that is specifically bound by a secondary binding agent.
In an eighth aspect, the invention provides a method for screening for the presence of a clinically relevant amount of Gram-positive bacteria in a donor tissue from a donor mammal for transfer to a recipient mammal, wherein the donor tissue is stored in a fluid, comprising contacting a sample of fluid with a set of binding agents, wherein the set of binding agents comprises binding agents that specifically bind to a Gram-positive bacterial antigen, and determining binding of the set of binding agents to the sample, wherein binding indicates the presence of a clinically relevant amount of Gram-positive bacteria in the donor tissue and no binding indicates the absence of a clinically relevant amount of Gram-positive bacteria in the donor tissue.
In certain embodiments of the eighth aspect of the invention, the sample is treated prior to or concomitantly with contacting the sample with the set of binding agents. Preferably, the treatment exposes a binding site for the binding agent on the Gram-positive bacterial antigen. In certain embodiments, the donor tissue determined to have an absence of a clinically relevant amount of bacteria is transferred to a recipient mammal. Preferably, the donor mammal and the recipient mammal are of the same species. In certain preferred embodiments, the binding agents that specifically bind to a Gram-positive bacterial antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-positive bacterial antigen. In certain embodiments, the binding agents that specifically bind to a Gram-positive bacterial antigen comprise an antibiotic, wherein the antibiotic is not vancomycin. Preferably, the binding agents that specifically bind to a Gram-positive bacterial antigen specifically bind to the lipotechoic acid structure of the Gram-positive bacteria.
In various embodiments of the eighth aspect of the invention, the binding agents that specifically bind to a Gram-positive bacterial antigen are detectably labeled with a reporter molecule. Preferably, the reporter molecule is a molecule with enzymatic activity, a radio-labeled molecule, a fusion molecule, a fluorogenic molecule, a metal sol, a particle, a chromatic molecule, or a molecule that is specifically bound by a secondary binding agent.
In a ninth aspect, the invention provides a method for screening for the presence of a clinically relevant amount of Gram-negative bacteria in a donor tissue from a donor mammal for transfer to a recipient mammal, wherein the donor tissue is stored in a fluid, comprising contacting a sample of the fluid with a set of binding agents, wherein the set of binding agents comprises binding agents that specifically bind to a Gram-negative bacterial antigen, and determining binding of the set of binding agents to the sample, wherein binding indicates the presence of a clinically relevant amount of Gram-negative bacteria in the donor tissue and no binding indicates the absence of a clinically relevant amount of Gram-negative bacteria in the donor tissue.
In certain embodiments of the ninth aspect of the invention, the sample is treated prior to or concomitantly with contacting the sample with the set of binding agents. Preferably, the treatment exposes a binding site for the binding agent on the Gram-negative bacterial antigen. In certain embodiments, the donor tissue determined to have an absence of a clinically relevant amount of Gram-negative bacteria is transferred to a recipient mammal. Preferably, the donor mammal and the recipient mammal are of the same species. In certain preferred embodiments, the binding agents that specifically bind to a Gram-negative bacterial antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-negative bacterial antigen. In certain embodiments, the binding agents that specifically bind to a Gram-negative bacterial antigen comprise a molecule selected from the group consisting of a Limulus anti-lipopolysaccharide factor (LALF), a lipopolysaccharide binding protein (LBP), a bactericidal/permeability-increasing protein (BPI), and an antibiotic, such as polymixin or bacitracin. Preferably, the binding agents that specifically bind to a Gram-negative bacterial antigen specifically bind to the lipopolysaccharide structure of the Gram-negative bacteria.
In various embodiments of the ninth aspect of the invention, the binding agents that specifically bind to a Gram-negative bacterial antigen are detectably labeled with a reporter molecule. Preferably, the reporter molecule is a molecule with enzymatic activity, a radio-labeled molecule, a fusion molecule, a fluorogenic molecule, a metal sol, a particle, a chromatic molecule, or a molecule that is specifically bound by a secondary binding agent.
In a tenth aspect, the invention provides a kit for screening for the presence of a clinically relevant amount of bacteria in a donor tissue from a donor mammal for transfer to a recipient mammal, wherein the tissue is stored in a fluid, comprising a set of binding agents, wherein the set of binding agents comprises binding agents that specifically bind to a Gram-negative bacterial antigen and binding agents that specifically bind to a Gram-positive bacterial antigen, and a means for detecting binding of the set of binding agents to a sample of the fluid, wherein binding indicates the presence of a clinically relevant amount of bacteria in the donor tissue and no binding indicates the absence of a clinically relevant amount of bacteria in the donor tissue.
In certain embodiments of the tenth aspect of the invention, the kit further comprises a means for treating the sample, wherein the treatment exposes a binding site for the binding agent on the Gram-negative bacterial antigen or on the Gram-positive bacterial antigen. In certain embodiments of the tenth aspect of the invention, the donor tissue determined to have an absence of a clinically relevant amount of bacteria is transferred to a recipient mammal. Preferably, the donor mammal from which the tissue was obtained and the recipient mammal are of the same species. In certain preferred embodiments, the binding agents that specifically bind to a Gram-negative bacterial antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-negative bacterial antigen. In certain preferred embodiments, the binding agents that specifically bind to Gram-negative bacteria or an antigen thereof comprise a molecule selected from the group consisting of a Limulus anti-lipopolysaccharide factor (LALF), a lipopolysaccharide binding protein (LBP), a bactericidal/permeability-increasing protein (BPI), and an antibiotic, such as polymixin or bacitracin. Preferably, the binding agents that specifically bind to a Gram-negative bacterial antigen specifically bind to the lipopolysaccharide structure of the Gram-negative bacteria. In certain preferred embodiments, the binding agents that specifically bind to a Gram-positive bacterial antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-positive bacterial antigen. In certain embodiments, the binding agents that specifically bind to a Gram-positive bacterial antigen comprise an antibiotic, wherein the antibiotic is not vancomycin. Preferably, the binding agents that specifically bind to a Gram-positive bacterial antigen specifically bind to the lipotechoic acid structure of the Gram-positive bacteria.
In various embodiments of the tenth aspect of the invention, the binding agents that specifically bind to a Gram-negative bacterial antigen are detectably labeled with a first reporter molecule and the binding agents that specifically bind to a Gram-positive bacterial antigen are detectably labeled with a second reporter molecule. In certain embodiments, the first reporter molecule and the second reporter molecule are the same. In another embodiment, the first reporter molecule and the second reporter molecule are not the same. Preferably, one or both of the first reporter molecule and the second reporter molecule is a molecule with enzymatic activity, a radio-labeled molecule, a fusion molecule, a fluorogenic molecule, a metal sol, a particle, a chromatic molecule, or a molecule that is specifically bound by a secondary binding agent.
In an eleventh aspect, the invention provides a kit for screening for the presence of a clinically relevant amount of Gram-positive bacteria in a donor tissue from a donor mammal for transfer to a recipient mammal, wherein the donor tissue is stored in a fluid, comprising a set of binding agents, wherein the set of binding agents comprises binding agents that specifically bind to a Gram-positive bacterial antigen, and a means for detecting binding of the set of binding agents to a sample of the fluid, wherein binding indicates the presence of a clinically relevant amount of Gram-positive bacteria in the donor tissue and no binding indicates the absence of a clinically relevant amount of Gram-positive bacteria in the donor tissue.
In certain embodiments of the eleventh aspect of the invention, the kit further comprises a means for treating the sample, wherein the treatment exposes a binding site for the binding agent on the Gram-positive bacterial antigen. In certain embodiments, the donor tissue determined to have an absence of a clinically relevant amount of Gram-positive bacteria is transferred to a recipient mammal. Preferably, the donor mammal and the recipient mammal are of the same species. In certain preferred embodiments, the binding agents that specifically bind to a Gram-positive bacterial antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-positive bacterial antigen. In certain embodiments, the binding agents that specifically bind to a Gram-positive bacterial antigen comprise an antibiotic, wherein the antibiotic is not vancomycin. Preferably, the binding agents that specifically bind to a Gram-positive bacterial antigen specifically bind to the lipotechoic acid structure of the Gram-positive bacteria.
In various embodiments of the eleventh aspect of the invention, the binding agents that specifically bind to a Gram-positive bacterial antigen are detectably labeled with a reporter molecule. Preferably, the reporter molecule is a molecule with enzymatic activity, a radio-labeled molecule, a fusion molecule, a fluorogenic molecule, a metal sol, a particle, a chromatic molecule, or a molecule that is specifically bound by a secondary binding agent.
In a twelfth aspect, the invention provides a kit for screening for the presence of a clinically relevant amount of Gram-negative bacteria in a donor tissue from a donor mammal for transfer to a recipient mammal, wherein the donor tissue is stored in a fluid, comprising a set of binding agents, wherein the set of binding agents comprises binding agents that specifically bind to a Gram-negative bacterial antigen, and a means for detecting binding of the set of binding agents to a sample of the fluid, wherein binding indicates the presence of a clinically relevant amount of Gram-negative bacteria in the donor tissue and no binding indicates the absence of a clinically relevant amount of Gram-negative bacteria in the donor tissue.
In certain embodiments of the twelfth aspect of the invention, the kit further comprises a means for treating the sample, wherein the treatment exposes a binding site for the binding agent on the Gram-negative bacterial antigen. In certain embodiments, the tissue determined to have an absence of a clinically relevant amount of Gram-negative bacteria is transferred to a recipient mammal. Preferably, the donor mammal and the recipient mammal are of the same species. In certain preferred embodiments, the binding agents that specifically bind to a Gram-negative bacterial antigen comprise antibodies or antibody derivatives that specifically bind to a Gram-negative bacterial antigen. In certain preferred embodiments, the binding agents that specifically bind to a Gram-negative bacterial antigen comprise a molecule selected from the group consisting of a Limulus anti-lipopolysaccharide factor (LALF), a lipopolysaccharide binding protein (LBP), a bactericidal/permeability-increasing protein (BPI), and an antibiotic, such as polymixin or bacitracin. Preferably, the binding agents that specifically bind to a Gram-negative bacterial antigen specifically bind to the lipopolysaccharide structure of the Gram-negative bacteria.
In various embodiments of the twelfth aspect of the invention, the binding agents that specifically bind to a Gram-negative bacterial antigen are detectably labeled with a reporter molecule. Preferably, the reporter molecule is a molecule with enzymatic activity, a radio-labeled molecule, a fusion molecule, a fluorogenic molecule, a metal sol, a particle, a chromatic molecule, or a molecule that is specifically bound by a secondary binding agent.
In various embodiments of the seventh, eighth, ninth, tenth, eleventh, and twelfth aspects of the invention, the donor tissue is preferably lung, heart, liver, skin, kidney, pancreas, spleen, or bone marrow.
In certain preferred embodiments of all of the above aspects of the invention, the set of binding agents is immobilized on a solid-phase support (e.g., a micro-titer plate).
In preferred embodiments of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, and twelfth aspects of the invention, the donor and/or recipient mammal is a human or a domesticated mammal.